An RNA‐binding atypical tropomyosin recruits kinesin‐1 dynamically to oskar mRNPs

Abstract

Localization and local translation of oskar mRNA at the posterior pole of the Drosophila oocyte directs abdominal patterning and germline formation in the embryo. The process requires recruitment and precise regulation of motor proteins to form transport‐competent mRNPs. We show that the posterior‐targeting kinesin‐1 is loaded upon nuclear export of oskar mRNPs, prior to their dynein‐dependent transport from the nurse cells into the oocyte. We demonstrate that kinesin‐1 recruitment requires the DmTropomyosin1‐I/C isoform, an atypical RNA‐binding tropomyosin that binds directly to dimerizing oskar 3′UTRs. Finally, we show that a small but dynamically changing subset of oskar mRNPs gets loaded with inactive kinesin‐1 and that the motor is activated during mid‐oogenesis by the functionalized spliced oskar RNA localization element. This inefficient, dynamic recruitment of Khc decoupled from cargo‐dependent motor activation constitutes an optimized, coordinated mechanism of mRNP transport, by minimizing interference with other cargo‐transport processes and between the cargo‐associated dynein and kinesin‐1.

Introduction

Within cells, organelles, diverse macromolecules and complexes depend on a small set of cytoskeleton‐associated motor proteins to achieve their proper distributions. Targeted delivery is ensured by cargo‐associated guidance cues that are responsible for recruiting the appropriate mechanoenzyme (Hirokawa et al, 2010). Such actively transported cargoes include mRNAs whose asymmetric localization and local translation within cells is essential for various cellular functions, such as migration, maintenance of polarity and cell fate specification (Medioni et al, 2012). In the case of messenger ribonucleoprotein (mRNP) particles, the guidance cues are the mRNA localization elements (LEs) that suffice to drive localization of the RNA molecule that contains them (Marchand et al, 2012). A few LEs, their RNA binding proteins (RBP) and the factors that link them to the mechanoenzyme have been well characterized (Dienstbier et al, 2009; Bullock et al, 2010; Dix et al, 2013; Niedner et al, 2014). In these cases, the entire localization process is driven by a single type of motor. Other mRNAs, such as Xenopus laevis Vg1 (Gagnon et al, 2013) and Drosophila melanogaster oskar (Clark et al, 2007; Zimyanin et al, 2008; Jambor et al, 2014), rely on the coordinated action of multiple motor proteins—cytoplasmic dynein and kinesin‐1 and kinesin‐2 family members—for their localization within developing oocytes.

oskar mRNA encodes the posterior determinant Oskar protein, which induces abdomen and germline formation in a dosage‐dependent manner in the fly embryo (Ephrussi & Lehmann, 1992). oskar mRNA is transcribed in the nurse cells of the germline syncytium and transported into the oocyte, similar to, for example, bicoid and gurken mRNAs (Ephrussi et al, 1991; Kim‐Ha et al, 1991). This first step of oskar transport is guided by a well‐described LE, the oocyte entry signal found in the 3′UTR of the mRNA (Jambor et al, 2014), which is thought to recruit the Egl–BicD–dynein transport machinery (Clark et al, 2007; Jambor et al, 2014). In the oocyte, oskar mRNA localization to the posterior pole is mediated by kinesin‐1 (Brendza et al, 2000; Zimyanin et al, 2008; Loiseau et al, 2010). This second step of oskar transport requires splicing of the first intron in the oskar pre‐mRNA, as oskar transcripts lacking all three introns (oskar Δi(1,2,3)) or just intron 1 (oskar Δi(1)) fail to localize (Hachet & Ephrussi, 2004). This splicing event results in assembly of the spliced localization element (SOLE) and deposition of the exon junction complex (EJC) on the mRNA (Ghosh et al, 2012). The EJC/SOLE constitutes a functional unit that is crucial for maintaining efficient kinesin‐1‐dependent transport of oskar mRNPs within the oocyte (Zimyanin et al, 2008; Ghosh et al, 2012), which is essential for proper localization of the mRNA to the posterior pole.

In a forward genetic screen, we identified a group of DmTm1 (formerly DmTmII) mutants (Tm1 ^gs) in which oskar mRNA accumulation at the posterior pole of the oocyte fails (Erdelyi et al, 1995; Fig 1A and B). Although the small amount of Oskar protein produced at the posterior pole is sufficient for embryo progeny of Tm1 ^gs homozygous females to form an abdomen and develop into adult flies, it is insufficient to induce primordial germ cell formation. Consequently, the Tm1 ^gs progeny are sterile, resulting in a so‐called grandchildless phenotype (Erdelyi et al, 1995). It was subsequently demonstrated that the microtubule‐mediated intra‐ooplasmic motility of oskar mRNPs is affected in Tm1 ^gs mutants (Zimyanin et al, 2008; Appendix Table S1), similar to what has been observed when kinesin‐1 is absent (Zimyanin et al, 2008). Although there is biochemical evidence that kinesin‐1 associates with oskar mRNPs (Sanghavi et al, 2013), what mediates the association of the motor with the mRNA, and where in the egg‐chamber this occurs, is not known.

Figure 1. Effect of Tm1 ^gs on oskar transport.

• A, B
  Localization of oskar mRNA in wild‐type (A) and Tm1 ^eg9/Tm1 ^eg9 (B) egg‐chambers.
• C
  Kymograph of oskMS2‐GFP mRNPs (cyan) travelling along polarity‐marked MTs (red, EB1 protein) in an ex vivo ooplasmic preparation. White dashed arrow shows the growing plus tip of the MT. Scale bars represent 1 s and 1 μm, respectively.
• D
  Distribution of oskMS2‐GFP mRNP runs towards plus (white) and minus ends (grey). Numbers within the bars indicate the number of runs. P‐value of chi‐squared test against wild type is indicated above each bar.
• E, F
  oskar mRNA (green) and Oskar protein (magenta) distribution in Tm1 ^eg1/Tm1 ^eg9 egg‐chambers expressing oskarMS2(6x) (E) or oskarMS2(6x) and Khc₄₀₁–MCP (F).
• G
  oskar mRNA (magenta) distribution in Tm1 ^eg1/Tm1 ^eg9 egg‐chambers rescued with EmGFP‐Tm1‐I (green).

Data information: Scale bars in (A, B and E–G) represent 50 μm.

Here, we demonstrate that DmTm1‐I/C, which consists mainly of low‐complexity sequences and a C‐terminal short tropomyosin superfamily domain (Cho et al, 2016), is an RNA‐binding tropomyosin that recruits kinesin heavy chain (Khc) to oskar mRNA molecules upon their nuclear export in the nurse cells. We show that Khc recruitment to oskar RNPs is transient and dynamic and that this dynamic recruitment depends on the presence of DmTm1‐I/C. Our data indicate that during mid‐oogenesis, the EJC/SOLE triggers kinesin‐1 activity, which drives localization of oskar mRNA to the posterior pole of the oocyte.

Results

Tm1‐I/C maintains proper levels of kinesin‐1 on oskar mRNA

To obtain mechanistic information regarding the motility defect in Tm1 ^gs oocytes, we developed an ex vivo assay that allows co‐visualization of MS2‐tagged oskar mRNPs and polarity‐marked microtubules (MTs) in ooplasm and determination of the directionality of oskar mRNP runs (Figs 1C and EV1A, and Video EV1), thus giving insight into the identity of the motor(s) affected by the Tm1 ^gs mutations. Using this assay, we found that in wild‐type ooplasm, plus‐end‐directed runs of oskMS2 RNPs dominated about two to one over minus‐end‐directed runs (Fig 1D). Plus‐end dominance was lost both in ooplasm lacking Khc and in extracts prepared from Tm1 ^gs‐mutant oocytes (Fig 1D). This indicates that plus‐end‐directed, Khc‐mediated motility is selectively compromised in the Tm1 ^gs mutants. The remaining plus‐end‐directed runs might be due to residual kinesin‐1 activity, to other plus‐end‐directed kinesins, or to cytoplasmic dynein, which has been shown to mediate the bidirectional random walks of mRNPs along MTs (Soundararajan & Bullock, 2014). To test whether the loss of Khc activity might be the cause of oskar mislocalization in Tm1 ^gs oocytes, we tethered a minimal Khc motor, Khc₄₀₁ (Sung et al, 2008; Telley et al, 2009), to the MS2‐tagged oskar mRNPs. Tethering of Khc₄₀₁–MCP to oskMS2 restored the plus‐end dominance of oskar mRNP runs (Fig 1D), as well as localization of oskar mRNA (Fig 1E and F), indicating that in Tm1 ^gs mutants a loss of kinesin‐1 activity might be the cause of oskar mislocalization.

Figure EV1. Ex vivo RNP motility and colocalization assays.

• A
  Schematic of ex vivo ooplasmic preparation imaged with TIRF microscopy.
• B
  Distribution of oskMS2‐GFP mRNP runs towards plus (white) and minus ends (grey). Black boxes indicate the fraction of RNP runs to which no polarity could be assigned (n.d.). Numbers within the bars indicate the number of runs.
• C, D
  Speed (C) and displacement (D) of oskMS2‐GFP mRNP runs towards plus and minus ends of MTs. Bars indicate wild‐type (green), Tm1 ^eg1/Tm1 ^eg9 (orange), Khc‐null (white) and Tm1 ^eg1/Tm1 ^eg9 + Khc₄₀₁ ooplasms (light blue), from left to right. The parameters are statistically not different from those determined in the wild‐type control (P > 0.05), with exception of the plus‐end‐directed transport length observed in Tm1 ^eg1/Tm1 ^eg9 extracts (*P = 1.4 × 10⁻⁴, pairwise Mann–Whitney U‐test). Runs of undetermined polarity were excluded from testing. Number of runs analysed is indicated in panel (B). The bottom and the top of the box represent the first and third quartiles, the thick horizontal lines indicate the data median. Whiskers show the data range excluding outliers, which are represented by dots.
• E, F
  Observed (points) and estimated random (coloured ribbons) colocalization of oskMS2‐mCherry mRNPs with different GFP fusion proteins (E) in different ooplasms (F) as a function of colocalization window size. MCP indicates MCP‐EGFP which, like MCP‐mCherry, can bind to MS2 loops. Numbers indicate the number of particle clusters (160 mRNPs in each) and the number of preparations (in brackets) analysed.
• G, H
  Difference between the observed and estimated random colocalization values. Horizontal black line above the graphs indicates the clipping point and regime when random colocalization dominates the difference values (H). For comparison of colocalization levels, the data points lying midway between zero distance and the onset of the random dominated regime (clipping point) were selected (at 200 nm, indicated by arrowheads). 0 indicates that colocalization values observed in Tm1 ^eg1/Tm1 ^eg9‐mutant ooplasms using 100‐nm colocalization window are not different from zero (P > 0.05, one‐sample t‐test).

Data information: (E–H) Error bars represent 95% confidence intervals.

The DmTm1 locus encodes at least 17 different transcripts and 16 different polypeptides (Appendix Fig S1A). By performing semi‐quantitative RT–PCR analysis, we found that the transcripts of Tm1‐C, Tm1‐I and Tm1‐H are selectively missing or their amount is greatly reduced in Tm1 ^eg1 and Tm1 ^eg9 homozygous ovaries, respectively (Appendix Fig S1B and C). Furthermore, an EmGFP‐Tm1‐I transgene expressed in the female germline rescued oskar mislocalization (Fig 1G) and the consequent grandchildless phenotype of Tm1 ^gs mutants (all female progeny (n > 20) contained at least one ovary with developing egg‐chambers). This indicates that the Tm1‐I/C isoform is essential for oskar mRNA localization.

To determine whether the reduction in Khc‐dependent oskar mRNP motility in Tm1 ^gs oocytes is due to insufficient kinesin‐1 recruitment or to insufficient motor activity, we analysed the composition of oskar mRNPs in ooplasms of flies co‐expressing either Khc‐EGFP or EmGFP‐Tm1‐I and oskMS2‐mCherry, ex vivo (Videos EV2 and EV3). We first performed an object‐based colocalization analysis of single snapshot images corrected for random colocalization (Fig EV1E–H). This revealed that both Khc‐EGFP (Fig 2A and Video EV2) and EmGFP‐Tm1‐I (Fig 2B and Video EV3) are recruited to a small but significant fraction of oskMS2‐mCherry mRNPs (1–4%; Fig 2C), indicating that the two proteins are components of oskar transport particles (Fig 2C). Furthermore, the association of Khc‐EGFP with oskMS2‐mCherry mRNPs was significantly reduced (two‐ to four‐fold) in Tm1 ^gs‐mutant ooplasms (Figs 2D and EV1H), indicating that the observed motility defects in Tm1 ^gs‐mutant oocytes are due to insufficient kinesin‐1 recruitment to oskar mRNPs.

Figure 2. Composition of oskar mRNPs ex vivo .

• A–B″
  Colocalization of oskMS2‐mCherry (red, A, B) (A′, B′) with Khc‐EGFP (green, A) (A′) or with EmGFP‐Tm1‐I (green B) (B′) in ex vivo ooplasmic preparations. Scale bars represent 5 μm.
• C
  Fraction of oskMS2‐MCP‐mCherry mRNPs located non‐randomly within a 200 nm distance of one of the indicated GFP‐tagged protein particles in ex vivo ooplasmic preparations. MCP indicates MCP‐EGFP which, like MCP‐mCherry, can bind to MS2 loops. Staufen (Stau) is a dsRNA binding protein and bona fide partner of oskar mRNA (St Johnston et al, 1991, 1992). All values are significantly different from zero (P < 10⁻³, one‐sample t‐test).
• D
  Fraction of oskMS2‐mCherry mRNPs colocalizing (max. 200 nm) non‐randomly with Khc‐EGFP particles in wild‐type and Tm1 ^eg1/Tm1 ^eg9 ooplasms in the presence of two (white) or one (grey) copy of endogenous Khc.

Data information: (C, D) P‐values of two‐sample t‐tests are indicated above the relevant bar pairs. Numbers indicate the number of particle clusters (160 mRNPs in each) and the number of preparations (in brackets) analysed. Error bars represent 95% confidence intervals.

In a complementary approach, we analysed Khc and Tm1‐I colocalization with oskMS2‐EGFP in consecutive images of entire time series. This assay estimates the probability of random colocalization in a different manner (Fig EV2A). For the analysis, we made use of flies expressing endogenously tagged Khc and Tm1‐I/C, in which virtually all molecules of interest are labelled (Fig 3A and B; Appendix Fig S1C and G). This revealed that in Khc ^mKate2 homozygous ooplasmic extracts, nearly 50% of motile oskMS2‐EGFP mRNPs are associated with Khc during at least half of the recorded trajectories (Fig 3C). As this value is close to the proportion of plus‐end‐directed runs (65%)—not all of which are mediated by Khc (Fig 1D)—and the fraction of Khc‐positive mRNPs is proportional to the amount of labelled Khc (Appendix Fig S1G), we assume that the rate of false negative detection is low in this analysis. In contrast to the high degree of association of Khc with motile mRNPs, only ~15% of non‐motile oskar particles were found to be in complex with Khc during their trajectories in the same analysis. The fact that at any given moment most oskar particles are stationary (Video EV1; Appendix Table S1; Zimyanin et al, 2008; Ghosh et al, 2012; Gaspar et al, 2014) indicates that, in accordance with the analysis of snapshot images (Fig 2D), the majority of oskar mRNPs are not in complex with Khc in wild‐type oocytes.

Figure EV2. Ex vivo temporal colocalization assay.

1. Schematics of temporal colocalization. oskarMS2 mRNPs were tracked in a manually assisted way. The mRNP particle outlines during a trajectory were used as a mask to measure the fluorescence of the fluorescently tagged protein molecules (e.g. Khc‐mKate2). The measured mean signal intensities of the assayed protein were compared to a threshold during every frame of the trajectory to determine colocalization of the protein of interest and the tracked mRNP (occupancy). These threshold values were determined as the lower 10^th percentile of the mean signal intensity distribution of all observed assayed protein “particles” (not exclusively those under the mask) in heterozygous extracts. None of the threshold values of a given protein molecule determined in individual extracts differed by more than 10% of their mean. The mean threshold value was used for a given protein both for hetero‐ and homozygous extracts. The sum of the occupancies divided by the total number of frames in the trajectory defined a single coverage value, which is plotted in Fig 3C–E. The probability of random occurrence of a coverage sequence was determined by calculating the corresponding frequency of a binominal distribution (Pr). As a “success probability in each trial” (P) measure, we used the estimated random colocalization values of each protein observed at ˜250 nm max distance, since the particle masks had an average diameter of 250 nm: for Khc‐mKate2, mCherry‐Tm1 and GFP‐Staufen, these P‐values were 0.132, 0.077 and 0.08, respectively. We scored a colocalization event as highly confident when the calculated Pr value was smaller than 0.01 and the coverage was ≥ 0.5. The fraction of these events is indicated above the boxplots in Fig 3C–E.
2. Duration of motile (white) and non‐motile (grey) oskar mRNP trajectories. To avoid bias during the determination of high‐confidence association, a population of non‐motile RNPs was selected whose duration did not differ significantly (P > 0.05, pairwise Mann–Whitney U‐test) from that of the corresponding motile RNP population. The Tm1 ^eg1/Tm1 ^eg9 data were an exception, as in this case we observed a significant difference between the duration of motile and non‐motile trajectories; however, there was no significant difference between the Khc coverage of the two motility categories (P = 0.023, Fig 3C). Frame duration (temporal resolution) is indicated above the boxplots. Numbers below the boxplots indicate the number of trajectories and the number of ooplasms (in brackets) analysed. The bottom and the top of the box represent the first and third quartiles, the thick horizontal lines indicate the data median. Whiskers show the data range excluding outliers, which are represented by dots.

Figure 3. Dynamic composition of oskar mRNPs ex vivo .

• A, B
  Kymographs of oskMS2‐GFP mRNPs (green) associated with Khc‐mKate2 (A, red) and mCherry‐Tm1‐I/C (B, red) ex vivo. Arrows indicate motile RNPs in stable complex with Khc (A) or Tm1‐I/C (B), and the arrowheads point to non‐motile oskMS2‐RNPs showing no obvious accumulation of the tagged protein. Note that mCherry‐Tm1‐I was exposed twice as long as Khc‐mKate to obtain comparable red fluorescence signals. Scale bars represent 1 μm and 1 s, respectively.
• C–E
  Relative Khc‐mKate2 (C), mCherry‐Tm1‐I/C (D) and GFP‐Staufen (E) coverage of motile (white) and non‐motile (grey) oskMS2‐GFP trajectories. Numbers within the boxes indicate the number of trajectories and the number of ooplasms (in brackets) analysed. Percentages above the plots show the fraction of RNPs that were found stably and reliably associating with the indicated protein (for at least half of the duration of the trajectory, P < 0.01, binominal distribution; see also Fig EV2B). P‐values of pairwise Mann–Whitney U‐tests are indicated above the boxplots. The bottom and the top of the box represent the first and third quartiles, the thick horizontal lines indicate the data median. Whiskers show the data range excluding outliers, which are represented by dots.
• F
  Example kymograph of Khc molecules (red) associating with an oskMS2‐GFP mRNP (green) before and during its run. White arrowheads indicate two association events, and yellow arrow indicates the onset of motility. Scale bars represent 5 μm and 1 s, respectively.
• G
  Frequency of Khc‐mKate2 appearance on motile (white), before (motility‐primed, checked) and after (dotted) the onset of motility, and non‐motile (grey) oskMS2‐GFP trajectories. Fractions within the bars indicate the number of association events that lasted longer than a single frame over the total number of frames analysed. Indicated P‐values show results of pairwise Fisher's exact test. The Khc association frequency observed on RNPs before (checked) and during (dotted) their motility is not significantly different from wild‐type motile RNP controls (P > 0.01).

We also found that ~20% of oskar mRNPs are stably associated with mCherry‐Tm1‐I/C irrespective of their motility (Fig 3B and D). The observed low and nonlinearly scaling proportions of oskar mRNPs (Fig 3D) associated with the relatively dim mCherry‐Tm1‐I (Fig 3B) suggest that (in contrast to Khc‐mKate2; see above) the false negative detection rate in this analysis is rather high. Therefore, we cannot reliably determine the true extent of the association and whether one‐fifth or a greater proportion of oskar mRNPs are associated with Tm1‐I/C. Nevertheless, our data show that like Staufen (Fig 3E), Tm1‐I/C is a component of oskar mRNPs in the oocyte.

Finally, our examination of Khc association with RNPs in mutant extracts lacking Tm1‐I/C revealed that it is equally low in the motile and non‐motile mRNP populations and that it is considerably below that observed in the wild‐type control (Fig 3C). This confirms our analysis of snapshot images and demonstrates that Tm1‐I/C is required for proper loading of Khc on oskar mRNPs.

Kinesin‐1 associates dynamically with oskar RNPs

During stage 9 of oogenesis, half of all oskar mRNA molecules in the oocyte translocate to the posterior pole (Gaspar et al, 2014). Since only 15% of oskar RNPs are in complex with Khc at any given moment (Fig 3C), this implies that kinesin‐1 must dynamically redistribute within the RNP population. To assess the possibility of such dynamic recruitment, we measured the frequency of the kinesin‐1 and oskar association events (Fig 3F). We observed that motile mRNPs associate with a Khc signal about once every 5 s (~0.2/s; Fig 3G) in wild‐type ooplasm before and during their motion. This frequency decreased to ~0.1/s in the case of wild‐type, non‐motile RNPs and dropped to ~0.05/s when Tm1‐I/C was absent (Fig 3G). This observation, the low degree of Khc association we observe in Tm1 ^gs‐mutant ooplasm and the interaction of Tm1‐I/C with oskar mRNPs (Figs 2C and 3D) indicate that Tm1‐I/C acts in recruiting the kinesin motor to the mRNA. The failure of this recruitment explains the greatly reduced number of long, unidirectional—in particular the plus‐end‐directed—runs of oskar mRNPs in the absence of Tm1‐I/C (Fig 1D; Appendix Table S1; Zimyanin et al, 2008).

Tm1‐I/C is in complex with Khc and oskar mRNA and directly binds the oskar 3′UTR

If Tm1‐I/C is indeed responsible for Khc recruitment to oskar mRNPs, these molecules should be in complex with one another. To test this hypothesis, we performed immunoprecipitations from ovarian lysates. Similar to what has been reported previously in vitro (Veeranan‐Karmegam et al, 2016), we detected that Khc specifically co‐immunoprecipitated with the EmGFP‐Tm1‐I bait in vivo (Fig 4A). Also, we found Staufen but no other tested oskar RNP components (Bruno, Y14, BicD or dynein) in the eluate (Fig 4A). Although this bulk co‐immunoprecipitation analysis cannot resolve either the heterogeneity or the spatiotemporal distribution of such complexes, it shows that Tm1‐I/C forms complexes with kinesin‐1 and with Staufen that are very likely maintained by—not necessarily direct—protein–protein interactions.

Figure 4. Tm1‐I/C binds RNA and Khc.

• A
  Western blots of oskar mRNP components (Staufen, Bruno and Y14) and motor‐associated proteins (Khc, BicD, Dic) co‐immunoprecipitated with EmGFP‐Tm1‐I from ovarian lysates. Protein marker bands and their molecular weight in kDa are indicated.
• B
  Signal developed with oligo(dT)₂₅–Texas Red probes on GFP‐Trap_M beads binding EmGFP‐Tm1‐I or FlagMycGFP after denaturing washes. 594 nm reflection marks bead boundaries. Scale bars represent 5 μm.
• B′
  Quantification of mean oligo(dT) signal measured on beads. P‐value of a pairwise Mann–Whitney U‐test is indicated. The bottom and the top of the box represent the first and third quartiles, the thick horizontal lines indicate the data median. Whiskers show the data range excluding outliers, which are represented by dots.
• C
  qRT–PCR of EmGFP‐Tm1‐I and control Flag‐Myc‐GFP and bead alone (w ¹¹¹⁸) bound oskar and gapdh RNAs after stringent washes (mean ± s.e.m., N = 3, two‐sample t‐test).
• D
  Images of beads binding to EmGFP‐Tm1‐I (green) and DIG‐labelled in vitro‐transcribed RNA fragments (magenta). Scale bars represent 5 μm.
• E, F
  Mean DIG‐Cy5 fluorescence measured on beads capturing the RNA fragment, with or without UV cross‐linking, indicated below the charts. P‐values of pairwise Mann–Whitney U‐tests are indicated. In panel (F), none of the non‐cross‐linked samples differ significantly from the no‐RNA control (P > 0.05). Numbers below the plots indicate the number of beads analysed. The bottom and the top of the box represent the first and third quartiles, the thick horizontal lines indicate the data median. Whiskers show the data range excluding outliers, which are represented by dots.

Source data are available online for this figure.

In a screen to identify proteins bound directly to mRNAs in early Drosophila embryos, we isolated a non‐isoform‐specific Tm1 peptide (Sysoev et al, 2016). By immunoprecipitating EmGFP‐Tm1‐I from lysates of embryos exposed to 254‐nm UV light, we detected significantly more poly(A)+ RNAs cross‐linked to Tm1‐I/C than to the control under denaturing conditions (Fig 4B and B′), confirming the RNA binding activity of TM1‐I/C. qRT–PCR of cross‐linked mRNAs revealed oskar as a target of Tm1‐I/C (Fig 4C). To identify the region of oskar mRNA to which Tm1‐I/C binds, we incubated embryonic lysates expressing EmGFP‐Tm1‐I with exogenous digoxigenin‐labelled oskar RNA fragments and subjected them to UV cross‐linking. Immunoprecipitation allowed the recovery of the 3′UTR, but not other regions of oskar mRNA (Fig 4D and E). Truncated (Fig EV3A–C) and non‐dimerizing oskar 3′UTR (Fig 4F) bound to EmGFP‐Tm1‐I with greatly reduced affinity. These findings indicate that Tm1‐I/C is an RNA binding protein and that its efficient binding to oskar mRNPs requires an intact, dimerizing oskar 3′UTR.

Figure EV3. RNA/RNP binding property of Tm1‐I/C in vitro and in situ .

• A–C
  Mean DIG‐Cy5 fluorescence of GFP‐Trap_M beads in the presence of the indicated RNA fragments with and without UV cross‐linking. In the case of the FlagMycGFP negative control (B) and the truncated, non‐binding oskar 3′UTR fragments (C), non‐cross‐linking conditions were not tested. P‐values of pairwise Mann–Whitney U‐tests are indicated. Numbers indicate the number of beads analysed. The bottom and the top of the box represent the first and third quartiles, the thick horizontal lines indicate the data median. Whiskers show the data range excluding outliers, which are represented by dots.
• D, E
  Observed (points) and estimated random (coloured ribbons) colocalization of oskar mRNPs with Khc‐mKate2 (green) or EmGFP‐Tm1‐I (red) or with both fusion proteins (blue) in wild‐type oocytes (C) and nurse cells (D). Arrows indicate the maximal colocalization distance (250 nm) chosen to analyse oskar mRNP composition in Fig 5F–I. The expected value of independent interaction was calculated by taking the difference of the products of observed individual colocalizations (Khc or Tm1‐I) and of expected individual colocalizations. Numbers indicate the number of particle clusters (100 oskar mRNPs in each) and the number of egg‐chambers (in brackets) analysed. Error bars represent 95% confidence intervals.

To test whether Tm1‐I/C and Khc co‐exist in oskar mRNP complexes, we performed oskar in situ hybridization on EmGFP‐Tm1‐I‐rescued Tm1 ^gs1 egg‐chambers carrying one copy of the Khc ^mKate2 allele (Fig 5A–C′). We found that only small portions of oskar mRNPs colocalized with Khc‐mKate2 (~4.6%) or EmGFP‐Tm1‐I (~5.7%) in oocytes in situ (Fig 4D), similar to what we observed in our ex vivo colocalization analysis (Fig 2C). Interestingly, the portion of oskar mRNPs positive for both Khc‐mKate2 and EmGFP‐Tm1‐I (~5.1%) was almost 40% higher than the value expected from the amount of colocalization of the mRNA with each component alone (P = 10⁻⁴; Figs 5F and EV3D). This positive correlation between the presence of Tm1‐I/C and Khc in oskar transport particles indicates that in most cases when one of the molecules is part of an oskar mRNPs, the other molecule is present as well. In a similar analysis, we found that the EJC component Mago, although part of oskar mRNPs, did not exhibit such a positive correlation of colocalization with Tm1‐I on the mRNA (Fig EV4A and B).

Figure 5. Composition of oskar mRNPs in situ .

• A–C′
  Confocal image of a Tm1 ^eg9 homozygous egg‐chamber expressing EmGFP‐Tm1‐I (green) and Khc‐mKate2 (magenta). oskar mRNA labelled with osk1‐5 FIT probes (Hovelmann et al, 2014) is in yellow. (B′, C′) oskar mRNPs colocalizing with both EmGFP‐Tm1‐I and Khc‐mKate2. Colours indicate the maximal colocalization distance (C′). Panels (B–C′) represent the boxed regions in panel (A).
• D, E
  Localization of Khc‐mKate2 (green) and oskar mRNA (magenta) in wild‐type and Tm1 ^gs‐mutant nurse cells.
• F, G
  Fraction of oskar mRNPs colocalizing with Khc‐mKate (dark grey), EmGFP‐Tm1‐I (white) or both of these proteins (light grey) in the oocyte (F) or in the nurse cells (G) (max. colocalization distance is 250 nm). None of the values are significantly different from each other (one‐way ANOVA, P > 10⁻³). Horizontal dashed lines indicate the expected value of observing both proteins in an oskar mRNP if the interactions are independent (see Fig EV3C and D). Significance of the observed colocalization values versus the expected values is shown (one‐sample t‐test). Data obtained from nurse cells and oocytes were compared with pairwise t‐test.
• H
  Fraction of oskar mRNPs colocalizing with Khc‐mKate in wild‐type and Tm1 ^gs‐mutant nurse cells when half or all Khc molecules are labelled (as indicated above the graph). P‐values of pairwise t‐tests are indicated.
• I
  Fraction of oskar mRNPs colocalizing with free mCherry in wild‐type nurse cells used as negative control. The measured fraction (˜1.6%) is significantly different from zero (one‐sample t‐test). All other measured colocalization values are significantly different from this negative control (P < 0.001, one‐way ANOVA).

Data information: (F–I) Numbers indicate the number of particle clusters (100 oskar mRNPs in each) and the number of egg‐chambers (in brackets) analysed. Error bars represent 95% confidence intervals. All values (F–I) are significantly different from zero (P < 10⁻³, one‐sample t‐test). Scale bars represent 20 μm (A) and 5 μm (B–E).

Figure EV4. Composition of oskar mRNPs in situ .

• A–A″
  Confocal image of a wild‐type egg‐chamber expressing GFP‐Mago (green, A′) and mKate2‐Tm1‐I (magenta, A”). oskar mRNA labelled with osk1‐5 FIT probes is in yellow.
• B
  Fraction of oskar mRNPs colocalizing with GFP‐Mago (green), mKate2‐Tm1‐I (orange), or both of these proteins (light grey) in the oocyte or in the nurse cells (max. colocalization distance is 250 nm). Horizontal dashed lines indicate the expected value of observing both protein in an oskar mRNP if the interactions are independent (see legend to Fig EV3D and E). Significance of the observed colocalization values versus the expected values is shown. In the nurse cells, GFP‐Mago and mKate2‐Tm1‐I appear to be independent (P > α = 0.001, one‐sample t‐test), whereas in the oocyte the presence of the two proteins on oskar mRNPs appears to anti‐correlate.
• C, C′
  Example confocal (C) and gated STED image (C′) of EmGFP‐Tm1‐I‐expressing nurse cells. EmGFP‐Tm1‐I is in magenta, and oskar mRNA is in green.
• D
  Difference between observed and estimated random colocalization of oskar mRNPs with oskMS2(10x)‐MCP‐GFP (black), EmGFP‐Tm1‐I (orange), Khc‐EGFP (blue) and Ketel‐GFP (importin‐β, red) in STED images. At 100 nm maximal distance, oskar mRNPs barely colocalize with the negative control Ketel‐GFP (P = 0.012, one‐sample t‐test versus zero). All other colocalization values are significantly different from that observed for Ketel‐GFP (P < 0.005, two‐sample t‐test).
• E
  Fraction of Khc‐EGFP‐positive oskar RNPs in the indicated nurse cells. P‐values of two‐sample t‐tests against wild type are indicated.
• F, G
  Mean density of detected oskar mRNA (dark grey), GFP‐Mago (green), mKate2‐Tm1‐I (orange) and Khc‐mKate2 (blue) in the nurse cell nuclei, cytoplasm and the ooplasm (F) and in the nurse cells under the conditions tested (G). All values are significantly different from zero (P < 0.05, one‐sample t‐test). Khc is considered to be a ubiquitous cytoplasmic protein, and therefore, we took its nuclear density as an estimate of the errors of the analysis (e.g. false positive recognition and uncertainties during definition of the nucleo‐cytoplasmic border). All other nuclear density values are significantly different from that of Khc‐mKate2 (F, P < 0.05, two‐sample t‐test). None of the Khc‐mKate2 and oskar mRNA density values are significantly different in the appropriate pairwise comparisons (G, P > 0.05). Numbers in the bars show the number of samples analysed.
• H
  Fraction of nuclear oskar mRNPs colocalizing with mKate2‐Tm1‐I (orange), GFP‐Mago (green) or Khc‐mKate2 (blue). The observed Tm1‐I/C‐ or Khc‐positive mRNPs are not different from zero (P‐values of one‐sample t‐tests are indicated, α = 0.01).
• I
  Fraction of Khc‐mKate2‐positive oskar mRNPs containing the full‐length mRNA (wild type) or only the oskar 3′UTR in the nurse cells. oskar mRNA was detected using a set of 15 singly labelled probes against the oskar 3′UTR.
• J
  Mean density of the detected Khc‐mKate2 (blue) and mRNPs labelled by the oskar 3′UTR probe set (grey) in the nurse cells (N = 5 each).

Data information: Numbers in the bars indicate the number of particle clusters (number of egg‐chambers) analysed. Error bars represent 95% confidence intervals. Scale bars represent 50 μm (A″) and 1 μm (C′).

Kinesin‐1 is recruited by Tm1‐I/C to oskar upon nuclear export

In the course of our FISH colocalization analysis, we noted that Khc‐mKate2 and EmGFP‐Tm1‐I colocalized with oskar mRNPs not only in the oocyte, but also in the nurse cell cytoplasm to the same extent (Figs 5A–B′ and G, and EV3E), indicating that the Khc recruiting machinery is already operational in the nurse cells. To test whether this colocalization of Khc with oskar mRNA also requires Tm1‐I/C, we analysed Khc‐mKate2 association with endogenous oskar mRNA in Tm1 ^gs‐mutant nurse cells (Fig 5D and E). We found an almost twofold reduction in Khc‐positive oskar mRNPs in the absence of Tm1‐I/C when a single Khc allele was labelled (Fig 5H). When all Khc molecules were fluorescently tagged, we also detected a significant difference in Khc association in Tm1 ^gs‐mutant and wild‐type nurse cells, although we did not observe the almost twofold increase in Khc‐positive oskar mRNPs in the wild‐type controls that we observed in case of the Tm1 ^gs mutant (Fig 5H). This observation highlights the possible limitation of our colocalization analysis when crowding of at least one of the objects occurs (Fig 5D and E). Although STED super‐resolution microscopy further increased crowding by resolving the confocal objects (Fig EV4C, C′ and G), it confirmed that both Khc‐EGFP and EmGFP‐Tm1‐I are recruited to oskar mRNPs (Fig EV4D). Moreover, it reinforced our finding that Khc‐EGFP association with oskar mRNPs in the nurse cells is greatly reduced in the absence of Tm1‐I/C (Fig EV4E).

Fluorescently tagged Tm1‐I, similar to Khc‐mKate, localized diffusely in the cytoplasm and, unlike other tropomyosins, did not accumulate on actin structures in vivo in the egg‐chamber (Fig 6C–D′)—although it was shown to bind microfilaments in vitro (Kim et al, 2011). In contrast, Tm1‐I accumulated at the posterior pole of the oocyte (Cho et al, 2016; Fig EV5A, C′ and E–G). Interestingly, we also detected the fluorescent Tm1‐I signal in the nurse cell nuclei (Figs 6A, EV4F and EV5B); however, in contrast to GFP‐Mago, we did not find evidence that nuclear Tm1‐I/C associates with oskar transcripts (Fig EV4H).

Figure 6. Accumulation of oskar mRNPs around the NE.

• A–C
  Localization of EmGFP‐Tm1‐I (A), oskar mRNA (B) and Khc (C) around the nurse cell nuclear envelope (magenta, WGA staining).
• D–F
  Mean distribution profile of Khc (D) mKate2‐Tm1‐I, EmGFP‐Tm1‐I (E) and oskar mRNA (F) around the NE of nurse cells (marked with a dotted vertical line). Genotypes are indicated as follows: wild‐type control with solid black line (D–F), oskar RNA null with solid grey line (D, E) and Tm1 ^eg9/Tm1 ^eg9 with dotted black line (D, F). Lines indicate mean and 95% confidence intervals. We analysed the accumulation of mKate2‐Tm1‐I instead of EmGFP‐Tm1‐I due to unexpected GFP expression from the oskar ⁰ allele.
• G
  Khc accumulation around the NE. To calculate accumulation, the signal intensity measured at the position of the peak observed in the wild‐type control (D, arrowhead, 356 ± 17.6 nm away from NE) was divided by the signal intensity 2 s.d. away (D, arrow, at 356 + 2*410 nm). P‐values of pairwise Mann–Whitney U‐tests against wild‐type control or Tm1 ^gs rescued with EmGFP‐Tm1‐I are indicated above the boxplots. Numbers indicate the number of nuclei and the number of egg‐chambers (in brackets) analysed. The bottom and the top of the box represent the first and third quartiles, the thick horizontal lines indicate the data median. Whiskers show the data range excluding outliers, which are represented by dots.
• H–K
  Khc accumulation around the NE of nurse cells over‐expressing EmGFP‐Tm1‐I within Tm1 ^eg9/Tm1 ^eg9 (H), and oskar RNA‐null egg‐chambers (I) expressing either the oskar 3′UTR (J) or oskar Δi(1,2,3) (K).

Data information: Scale bars are 10 μm (A–C) and 5 μm (H–K).

Figure EV5. Localization of Tm1‐I/C.

• A
  Localization of EmGFP‐Tm1‐I expressed within the female germline.
• B
  Intensity profile of EmGFP‐Tm1‐I along the dashed arrow in (A). Cellular compartments are indicated above the curve. The non‐EmGFP‐Tm1‐I‐expressing follicle cells serve as a background reference.
• C–D′
  Localization of mCherry‐Tm1 (magenta) relative to actin (green) within egg‐chambers. (D and D′) High‐magnification imaging of nurse cells reveals dim actin fibres surrounding the nuclei. However, mCherry‐Tm1 puncta do not appear to co‐align with these fibres. An infolding of the NE (D′, arrows) appears as an enrichment of mCherry‐Tm1 and actin inside the nucleus.
• E, F
  Immunostaining of EmGFP‐Tm1‐I‐expressing (E–E″) and wild‐type (F) PFA‐fixed egg‐chambers with pan‐Tm1 antibody. The Tm1 antibody staining (green) shows a signal distribution similar to that of EmGFP‐Tm1‐I (magenta) within oocytes; however, Tm1‐I localization around the nurse cell NE is barely recognizable in PFA‐fixed mid‐oogenetic egg‐chambers (E′ and F).
• G–I
  In heat‐fixed specimen, on the other hand, we could detect a faint perinuclear accumulation of the pan‐Tm1 antibody signal in wild‐type (G) but not in Tm1 ^gs‐mutant (H) stage 8–10 nurse cells. The subtle accumulation we could quantify (I: N_wt = 23, N_Tm1gs = 18). Lines indicate mean and 95% confidence intervals.

Data information: Scale bars represent 50 μm (A, C, E–H) and 10 μm (D).

The most prominent localization pattern of the fluorescently tagged Tm1‐I was its enrichment around the nuclear envelope (NE) in the nurse cells (Figs 6A and EV5; Cho et al, 2016; Veeranan‐Karmegam et al, 2016), similar to that of oskar mRNA (Little et al, 2015; Fig 6B). We also detected perinuclear enrichment of endogenous Tm1‐I/C on immunolabelled wild‐type specimens (Fig EV5G and I), although it was less pronounced due to the high aspecific signal in the nurse cell cytoplasm (Fig EV5H). We observed that Khc also enriches around the nurse cell NE (Fig 6C), although to a lesser extent than the fluorescently tagged Tm1‐I/C or oskar mRNA detected by FISH. Our analysis of radial profiles of NEs counterstained by fluorescent lectins confirmed a slight but significant perinuclear accumulation of Khc in wild‐type nurse cells (Fig 6D and G), independent of the developmental age of the egg‐chamber (Appendix Fig S2A). This observed Khc accumulation around the NE required the presence of both oskar mRNA and Tm1‐I/C (Fig 6D and G). In contrast, oskar mRNA or mKate2‐Tm1‐I accumulation around the NE was not affected in Tm1 ^gs1 or oskar‐null mutant egg‐chambers, respectively (Fig 6E and F).

We noted that transgenic over‐expression of EmGFP‐Tm1‐I increased Khc recruitment to the NE substantially in the rescued Tm1 ^gs egg‐chambers (Fig 6G and H). There was a slight elevation in Khc accumulation in the absence of oskar RNA (Fig 6G and I), possibly reflecting the ability of the over‐expressed EmGFP‐Tm1‐I to bind Khc on its own, or in the presence of other, even non‐specific RNA targets. However, a substantial increase was only observed when an intact oskar 3′UTR, whether in endogenous oskar mRNA, transgenic non‐spliced, non‐localizing oskar Δi(1,2,3) or the oskar 3′UTR alone, was present (Fig 6G, J and K). Given that even in the absence of oskar mKate2‐Tm1‐I was enriched around the NE, these results not only confirm the instrumental role of Tm1‐I/C in the Khc recruitment process, but also indicate that kinesin‐1 loading on oskar mRNPs takes place if and only if mRNAs containing the oskar 3′UTR are available. Consistent with this, in the nurse cell cytoplasm of oskar RNA‐null egg‐chambers expressing the oskar 3′UTR and Khc‐mKate2, we detected an identical degree of Khc association with oskar mRNPs as observed in wild‐type control egg‐chambers (Fig EV4I and J).

Kinesin recruited by Tm1‐I/C is inactive

To address the functional consequences of the “super‐loading” of Khc at the NE upon EmGFP‐Tm1‐I over‐expression, we quantified the mean distribution of the intronless, non‐localizing oskar Δi(1,2,3) RNA (Hachet & Ephrussi, 2004; Ghosh et al, 2012) throughout stage 9 oocytes (Gaspar et al, 2014). This analysis showed that over‐expression of EmGFP‐Tm1‐I causes a substantial posterior‐ward shift of the non‐spliced oskar Δi(1,2,3) mRNA (Fig 7A, B and F). However, the rescue of localization was not complete as it still deviated substantially from the wild‐type control (Fig 7C and F). Furthermore, EmGFP‐Tm1‐I over‐expression did not promote posterior localization of an RNA consisting solely of the oskar 3′UTR (Fig 7D and F), although the posterior enrichment of Khc was not affected (Appendix Fig S2F and G). EmGFP‐Tm1‐I over‐expression also did not promote oskar mRNA localization in oocytes with reduced Khc levels (Fig 7E and F; Appendix Fig S2H), confirming the essential role of kinesin‐1 motor is this process. These observations indicate that a properly assembled EJC/SOLE is required to activate the oskar 3′UTR‐bound, Tm1‐I/C‐recruited kinesin‐1 within the oocyte during mid‐oogenesis.

Figure 7. Effects of EmGFP‐Tm1‐I over‐expression on oskar mRNA localization.

• A–E
  Mean oskar mRNA distribution (green, detected by conventional FISH) within oocytes in which oskar mRNA is substituted by oskar Δi(1,2,3) mRNA (A, B) and oskar 3′UTR mRNA (D) and (in addition) over‐express EmGFP‐Tm1‐I (B, D and E). Wild‐type control (C) and oocytes expressing Khc RNAi and EmGFP‐Tm1‐I (E). Magenta indicates standard deviation of the distribution of oskar mRNA. Scale bar is 10% of total oocyte length (C).
• F
  Position of the oskar mRNA centre of mass relative to the geometric centre of the oocyte (dotted horizontal line) along the anteroposterior (AP) axis. Posterior pole is the top of the chart. P‐values of pairwise Mann–Whitney U‐tests are indicated. Numbers indicate the number of oocytes analysed. The bottom and the top of the box represent the first and third quartiles, the thick horizontal lines indicate the data median. Whiskers show the data range excluding outliers, which are represented by dots.

Discussion

Loading of the appropriate transport machinery on mRNPs is critical to achieve correct localization and, consequently, localized translation of the transcript. Although mRNA transport has been extensively studied over the last two decades, the recruitment of plus‐end‐directed kinesin motors to mRNPs and their regulation remains poorly understood (Medioni et al, 2012).

Here, we have shown that the majority of kinesin‐1 motor associated with oskar mRNA is recruited by Tropomyosin1‐I/C, a non‐canonical RNA binding protein, which explains the mislocalization of oskar mRNA when Tm1‐I/C is lacking (Erdelyi et al, 1995; Veeranan‐Karmegam et al, 2016; and this study). We found that this recruitment occurs early in the cytoplasmic life of the mRNA, upon its nucleo‐cytoplasmic export, in the perinuclear cytoplasm of the nurse cells, where dimerization of oskar mRNA molecules via their 3′UTRs commences (Little et al, 2015). Our observations are consistent with an “ergonomic” kinesin‐loading machinery that becomes functional when and only when Tm1‐I/C binds the oskar 3′UTR in the nurse cell cytoplasm. The kinesin‐recruitment machinery is inefficient, as only a small portion of oskar mRNPs are kinesin‐bound even during the most active phase of oskar mRNA posterior‐ward transport in the oocyte. Such inefficiency may serve to prevent the sequestration of kinesin‐1 molecules by oskar mRNPs away from other cargoes requiring the motor for transport. On the other hand, the transient and dynamic binding and unbinding of the motor may enable transport of virtually all oskar mRNPs in a temporally coordinated fashion, thereby promoting localization of more than 50% of oskar mRNA to the posterior pole of the oocyte by the end of stage 9 (Gaspar et al, 2014). Additionally, as oskar mRNA is continuously transported from the nurse cells to the oocyte from the beginning of oogenesis (Kim‐Ha et al, 1991; Ephrussi & Lehmann, 1992; Jambor et al, 2014), the age of oskar mRNPs at the onset of posterior‐ward transport may vary between a few minutes and one/one and a half days. The dynamic recruitment of Khc may also guarantee that the oskar particles are equipped with transport‐competent motor molecules at any moment. It is also apparent from our data that a smaller amount of Khc is recruited to oskar mRNPs independent of Tm1‐I/C. However, the dynamic exchange of these Khc molecules appears to be rather slow and, most likely as a consequence, they mediate only a minuscule fraction of intra‐ooplasmic oskar transport—as inferred from the almost complete loss of motility in Tm1 ^gs mutants (Appendix Table S1)—highlighting the importance of Tm1‐I/C in kinesin loading.

The first step of oskar transport is mediated by cytoplasmic dynein (Clark et al, 2007; Jambor et al, 2014), which is presumably also recruited at the nurse cell NE, where we detected the accumulation of the RNA cargo adapter Egalitarian (Appendix Fig S2C; Navarro et al, 2004; Dienstbier et al, 2009) and the dynactin component dynamitin (Appendix Fig S2D; McGrail et al, 1995). Interestingly, the dynein apoenzyme did not enrich around the NE (Appendix Fig S2B and E), possibly because its association with oskar mRNPs instantly initiates their transport away from the NE into the oocyte. Within the oocyte, efficient posterior‐ward transport of oskar mRNA only takes place upon repolarization of the MT cytoskeleton during mid‐oogenesis (Theurkauf, 1994; Parton et al, 2011). Therefore, the activity of the kinesin‐1 recruited to oskar mRNPs must be regulated and coordinated with that of the dynein in response either to environmental changes (Gaspar et al, 2014; Burn et al, 2015) or to the developmental programme. Our data showing the failed or incomplete posterior localization of oskar 3′UTR and oskar Δi(1,2,3), respectively, indicate that the kinesin‐1 recruited to the oskar 3′UTR by Tm1‐I/C is inactive. Since the oskar coding sequence—with the exception of the SOLE—is dispensable for the localization process (Ghosh et al, 2012), we propose that during mid‐oogenesis, the spliced, EJC‐associated SOLE complex activates the oskar RNA‐bound kinesin‐1. Therefore, although the EJC/SOLE is necessary for proper oskar mRNA localization, it is not sufficient, as recruitment of the kinesin‐1 motor to oskar mRNPs is mediated by Tm1‐I/C, whose RNA binding scaffold is provided by the oskar 3′UTR. To our knowledge, oskar is the first mRNA and the first cargo of kinesin‐1 described where loading and activation of the motor are decoupled and an unproductive tug of war between two opposing motors is avoided by keeping one of the molecules in a long‐term stasis.

Although the underlying mechanisms of the Khc loading and activation processes remain cryptic, a recent study demonstrated that the deletion of the ATP‐independent MT‐binding site and the auto‐inhibitory IAK motif of Khc results in phenotypes consistent with a failure in motor activation and/or recruitment to oskar mRNPs (Williams et al, 2014). Moreover, it was shown parallel with our study that Tm1‐I/C is able to directly bind Khc in vitro and that this interaction depends on the ATP‐independent MT‐binding site (Veeranan‐Karmegam et al, 2016), indicating that Tm1‐I/C directly links kinesin‐1 to oskar mRNA. This unconventional mode of cargo binding via the unconventional cargo adapter Tm1‐I/C may result in incomplete release of kinesin from auto‐inhibition and thus explain the persistent inactivity of the kinesin‐1 upon its recruitment to the mRNA.

Tm1‐I/C is an atypical tropomyosin: it does not enrich around microfilaments in the female germline (Cho et al, 2016; Veeranan‐Karmegam et al, 2016; and this study), forms intermediate filament‐like structures (Cho et al, 2016) and directly binds to Khc in vitro (Veeranan‐Karmegam et al, 2016) and to RNA, recruiting kinesin‐1 dynamically to oskar mRNPs. Although Tm1‐I/C has a short tropomyosin superfamily domain in its C‐terminal moiety, most of the protein is composed of low‐complexity sequences (Cho et al, 2016). Such intrinsically disordered proteins (IDPs) are often constituents of RNA‐containing membraneless organelles, such as RNA granules (Kato et al, 2012), stress granules (Molliex et al, 2015), P granules (Elbaum‐Garfinkle et al, 2015) and nuage and germ granules (Nott et al, 2015). Furthermore, it has been shown that IDPs fold upon binding to their partners (Wright & Dyson, 2009). This may explain why Tm1‐I/C only recruits Khc to the NE in the presence of the oskar 3′UTR. Further dissection of the structure and the precise molecular functions of Tm1‐I/C and its binding partners will be crucial to determining the nature and the minimal number of features necessary for such unconventional yet vital kinesin‐1‐mediated localization of mRNA.

Materials and Methods

Fly stocks

The Tm1 ^eg1 and Tm1 ^eg9 (FBal0049223) mutants originating from the imprecise excision of the P‐element in Tm1 ^gs1 (FBal0049228) were used to study the role of Tm1 in oskar mRNA localization (Erdelyi et al, 1995). To remove oskar mRNA (oskar RNA null), a newly created osk ^attP,3P3GFP (oskar ⁰ in this manuscript) allele was used in homozygous form or in combination with another RNA‐null allele, osk ^A87 (FBal0141009). We observed no difference between the phenotype of osk ^attP,3P3GFP homozygotes and the osk ^attP,3P3GFP/osk ^A87 heterozygotes in our assays. The following oskar mRNA mutations and truncations were expressed in an oskar RNA‐null background: UASp‐osk.3′UTR (Filardo & Ephrussi, 2003; FBal0143291) and UASp‐oskar Δi(1,2,3) 5x BoxB (Ghosh et al, 2012; no protein coding function, FBal0291667). αTub67C::GFP ^m6 ‐Staufen (Schuldt et al, 1998; FBal0091177), UASp‐dmn‐GFP (Januschke et al, 2002; FBal0145074), αTub::Khc‐EGFP (Sung et al, 2008; FBal0230204), UASp‐GFP‐Mago (Newmark et al, 1997; FBal0063884) and Ketel‐GFP (Villanyi et al, 2008; FBal0244142) were used to visualize Staufen, dynamitin, Khc, Mago and importin‐β molecules. To express mCherry FP in the nurse cells, we used the TM3, P{sChFP} balancer chromosome (FBti0141181). The Khc ²⁷ protein‐null allele (FBal0101625) was used to halve Khc levels (heterozygous) or completely remove Khc from egg‐chambers developing from Khc ²⁷ homozygous germline clones; the UASp‐Khc RNAi Trip Line GL00330 (Staller et al, 2013) was used to knock down Khc levels. To label oskar mRNPs, we used the osk::oskMS2(10x) system together with hsp83::MCP‐EGFP (Zimyanin et al, 2008) or hsp83::MCP‐mCherry (a gift from L. Gavis) and UASp‐EB1‐mCherry (a gift from D. Brunner) to label the growing plus ends of MTs. For the Khc tethering experiment, we expressed osk::oskMS2(6x) (Lin et al, 2008; FBal0263509), as oskMS2(10x) is not translated at the posterior pole (Zimyanin et al, 2008). Expression of all UASp transgenic constructs was driven with one copy of oskar‐Gal4 (Telley et al, 2012; FBtp0083699), with the exception of the Khc RNAi line, where a second oskar‐Gal4 allele was introduced to boost the expression level of co‐expressed UASp‐EmGFP‐Tm1‐I. w ¹¹¹⁸ (FBal0018186) was used as the wild‐type control. All stocks were raised on normal cornmeal agar at 25°C. The generation of transgenic lines for this study is described in the Appendix Supplementary Materials.

Immunological techniques

Immunoprecipitation of EmGFP‐Tm1‐I and Flag‐Myc‐GFP (as control) from ovarian lysates was carried out as described (Ghosh et al, 2014). Eluates were tested in Western blot analysis probing with anti‐Khc (1:5,000; Cytoskeleton), anti‐Staufen (1:1,000) (Krauss et al, 2009), anti‐Bruno (1:5,000) (Chekulaeva et al, 2006), anti‐Y14 (1:2,500) (Hachet & Ephrussi, 2001), anti‐BicD (2:100, DHSB clone #1B11 and 4C2), anti‐Dic (1:2,000; Millipore) and anti‐GFP (1:2,000; Millipore). The same antibodies and dilutions were used to detect RNAi knockdown efficiency and relative levels of Khc‐EGFP. To test Tm1‐I/C expression in ovarian lysates, a pan‐Tm1 antibody (1:1,000) (Cho et al, 2016) recognizing the tropomyosin domain shared by the Tm1 isoforms was used (a gift from D. Montell).

Khc, Tm1, Dic and Egl were visualized in heat‐fixed egg‐chambers (Gaspar et al, 2014) incubated overnight at 4°C in anti‐Khc (1:250), pan‐Tm1 (1:500) (Cho et al, 2016), anti‐Dic (1:1,000) or anti‐Egl (1:1,000) (Navarro et al, 2004) primary antibodies diluted in PBST (PBS + 0.1% Triton X‐100)/10% normal goat serum. Signal was developed by applying AlexaFluor 488‐ or Cy5‐conjugated anti‐rabbit or anti‐mouse secondary antibodies for 60 min at room temperature (RT) (1:1,000; Jackson ImmunoResearch). GFP autofluorescence was preserved by fixation for 20 min in 2% PFA/0.05% Triton X‐100 in PBS. To visualize F‐actin, samples were fixed in 2% PFA/0.05% Triton X‐100 in BRB80 (80 mM PIPES, 2 mM MgCl₂, 1 mM EGTA) and stained with phalloidin–FITC (1:100; Molecular Probes), and all washes were carried out in BRB80 + 0.1% Triton X‐100.

NE was counterstained by WGA‐TRITC (1:300; Life Sciences) for 60 min at RT. Samples were embedded in 80% glycerol + 2% N‐propyl‐gallate mounting medium.

RNA recovery after cross‐linking

To test whether Tm1‐I/C binds RNA directly, 0 to 2‐h‐old embryos expressing EmGFP‐Tm1‐I, EGFP or FlagMycGFP were collected and 1 J/cm² UV (λ = 254 nm) was applied to cross‐link nucleic acids with protein molecules at zero distance. Embryos were lysed in 10× RIPA buffer composed of low‐salt buffer (20 mM Tris–Cl, 150 mM KCl, 0.5 mM EDTA, pH = 7.5) containing strong detergents (1% Triton X‐100, 1% deoxycholate and 0.1% SDS), reducing agent (5 mM DTT) and 5× protease inhibitor cocktail (PIC; Roche), and crude debris was removed by 10‐min‐long centrifugation with 16,000 g at 4°C. Clarified lysates were diluted to 10 mg/ml with 10× RIPA buffer and then further diluted tenfold with low‐salt buffer. 1 mg protein of lysate (1 ml) was supplemented with 10 μl of GFP‐Trap_M beads (Chromotek), and immunoprecipitation was carried out at 4°C for 90 min. Beads were washed six times with high‐salt buffer (20 mM Tris–Cl, 500 mM NaCl, 1 mM EDTA, 0.1% SDS, 0.5 mM DTT, 1× PIC pH = 7.5) at room temperature for a total duration of 60 min (stringent washes). Alternatively, beads were washed twice with high‐salt buffer and then three times under denaturing conditions with 7.6 M urea and 1% SDS in PBS (Chromotek Application Note). RNA was then extracted from the washed beads, and qRT–PCR or FISH was performed on the beads with oligo(dT)25–Texas Red (Castello et al, 2012). FISH‐on‐beads experiments were imaged on a Leica SP8 with a 63× 1.4 NA oil‐immersion objective. Bead contours were captured by imaging the reflection of 594‐nm light, and oligo(dT)25 probes were excited with 594 nm. Primer sequences used during qRT–PCR are available upon request.

To analyse oskar mRNA fragments binding to EmGFP‐Tm1‐I, embryos were lysed in pre‐XL buffer (20 mM Tris–Cl, 150 mM KCl, 4 mM MgCl₂, 2× PIC, pH = 7.5) and cleared from debris, and lysate concentration was adjusted to 10 mg/ml. 0.2–0.8 ng/nucleotide DIG‐labelled RNA (1:10 ratio of labelled to unlabelled uracil) and 1 μl RiboLock (ThermoFisher Scientific) were added to 100 μl lysate. Mixtures were incubated for 20 min at RT prior to cross‐linking with 254‐nm UV light, 0.15 J/cm² energy. Subsequently, the lysate was diluted with an equal volume of low‐salt buffer supplemented with detergents, reducing agent and PIC (see above) and further diluted 1:5 with low‐salt buffer. 2 μl RiboLock and 5 μl GFP‐Trap_M beads were added per 500 μl volume, and immunoprecipitation and washes were carried out as described above. Beads were then labelled with anti‐GFP‐CF488A (1:1,000; Sigma‐Aldrich) and anti‐DIG‐HRP (1:500; Roche) PBST for 30 min. Anti‐DIG signal was developed using Cy5‐TSA amplification kit (PerkinElmer). Fluorescently labelled beads were mounted on slides in glycerol‐based mounting medium and were imaged with a Leica 7000 TIRF microscope using a 100× 1.46 NA oil‐immersion objective, 1.6× Optovar and epifluorescent illumination.

Fluorescent in situ hybridization

For conventional FISH, specimens were fixed in 2% PFA/0.05% Triton X‐100 in PBS for 2 h at RT. After fixation and washes in PBST, 2 μg/ml proteinase K was applied for 5 min at RT, followed by 5 min boiling at 92°C in PBS/0.05% SDS. The samples were then pre‐hybridized for 60 min at 65°C in hybridization buffer (HYBEC: 5× SSC, 15% ethylene carbonate, 50 μg/ml heparin, 0.1 mg/ml salmon sperm DNA, 0.05% SDS, pH = 7.0). The oskar cds (targeting nucleotides 1,442–1,841) and oskar 3′UTR (targeting 2,342–2,730) probes were diluted in HYBEC in 0.5 pg/nucleotide/ml concentration each. The oskar cds probes were directly labelled with either Atto‐488 (Atto‐tec) or AlexaFluor 555 (ThermoFisher Scientific), while the oskar 3′UTR was directly labelled with Atto‐633 (Atto‐tec). Hybridization was carried out at 65°C overnight and excess probe was removed by four washes at 65°C (HYBEC, HYBEC/PBST 1:1, 2× PBST, each 20 min) and one 20‐min‐long wash in PBST at RT. During conventional FISH, WGA‐FITC was applied to counterstain NEs. Samples were embedded in 80% glycerol + 2% N‐propyl‐gallate mounting medium.

To preserve GFP and mKate2 autofluorescence, forced intercalation (FIT) probe‐based RNA detection was performed (Hovelmann et al, 2014). Ovaries were fixed for 20 min in 2% PFA/0.05% Triton X‐100 in PBS, and they were washed twice for 10 min in IBEX (10 mM HEPES, 125 mM KCl, 1 mM EDTA, 0.3% Triton X‐100, pH = 7.7) after fixation. oskar mRNA was labelled with osk1‐5 LNA‐modified FIT probes (Hovelmann et al, 2014) in IBEX+ (IBEX supplemented with 15% ethylene carbonate, 50 μg/ml heparin and 10% 10‐kDa dextran sulphate) to a final concentration of 0.05 μM each. Samples were incubated at 42°C for 30 min and then briefly washed in IBEX and IBEX/BRB80 (1:1 mixture) at 42°C.

To detect oskar 3′UTR with single‐molecule FISH (smFISH), we used 15 different 3′UTR‐targeting probes labelled with a single Atto‐565‐ddUTP nucleotide using TdT (Appendix Table S2). smFISH was carried out similar to conventional FISH. Proteinase K digestion and heat denaturation of RNA secondary structures were omitted to preserve mKate2 fluorescence. The hybridization was performed at 37°C for 2 h using 5 nM/probe concentration.

Specimens were embedded in 79% TDE (η = 1.475; Staudt et al, 2007), which boosted the brightness of GFP and mKate2 by about twofold and of FIT probes four‐ to six‐fold compared to conventional glycerol‐based mounting media (data not shown).

Microscopy

Conventional laser scanning confocal microscopy was carried out using a Leica TCS SP8 microscope with a 63× 1.4 NA oil‐immersion objective. STED microscopy was performed on a Leica STED 3× microscope with a 100× 1.4 NA oil‐immersion objective and HyD time‐gated photodetectors. Acquired images were deconvolved with Huygens Professional (SVI) prior to analysis.

To minimize crosstalk of the two labels, GFP‐ and TO‐labelled FIT probes were excited with 470‐nm and 525‐nm lines of a white‐light laser source, respectively. Emission was recorded between 480 and 520 nm (GFP) and 525 and 575 nm (TO). For similar reasons, Atto‐565 and mKate2 were excited by 561‐nm and 610‐nm light and emitted fluorescence was detected between 565 and 585 nm and between 620 and 720 nm, respectively. Under these conditions, < 1% of recorded signal originated from crosstalk.

To stimulate emission of the GFP and TO dyes, a 592‐nm depletion doughnut‐shaped laser beam was used, with all power assigned to improve lateral resolution by about 2.5‐ to 3‐fold. Typically, a stack of seven slices was recorded (voxel size: 22 × 22 × 180 nm) and subsequently deconvolved with Huygens Professional. The middle slice was then subjected to object‐based colocalization analysis.

Ex vivo ooplasmic preparation

Crude ooplasm was obtained from living stage 9 oocytes expressing oskMS2(10x), MCP‐EGFP, oskGal4 and EB1‐mCherry for mRNP tracking or oskMS2(10x), MCP‐mCherry and a GFP‐tagged protein of interest for ex vivo colocalization analysis. Ovaries were dissected in BRB80 (80 mM PIPES, pH = 6.9, 2 mM MgCl₂, 1 mM EGTA). BRB80 was replaced with 1% IB (10 mM HEPES, pH = 7.7, 100 mM KCl, 1 mM MgCl₂, 1% 10‐kDa dextran), and ovaries were transferred onto silanized coverslips. Silanization was carried out with dichlorodimethylsilane (Sigma‐Aldrich) under vacuum for 1.5–2 h to obtain a slightly hydrophobic surface. A drop of Voltalef 10S oil (VWR) was placed next to the dissected ovaries, and individual ovarioles containing stage 9 egg‐chambers were pulled under oil with fine tungsten needles. There, the stage 9 egg‐chambers were isolated and the nurse cell compartment was carefully removed with needles. Using a gentle pulling force at the posterior pole of the created “oocyte sack” (the oocyte and surrounding follicle cells), the ooplasm was slowly released from anterior to posterior onto the coverslip surface. The level of surface hydrophobicity was critical: a hydrophilic surface bound oskar mRNPs aspecifically, blocking their motility, whereas it was impossible to create an ooplasmic streak on coverslips that were too hydrophobic. Such preparations were imaged on a Leica 7000 TIRF microscope with a 100× 1.46 NA oil objective and 1.6× Optovar. Images were collected simultaneously for 32 s with a Photometrics Evolve 512 EM CCD camera with 140 nm lateral resolution. We observed no decline in RNP motility and MT dynamics within the first 60 min (data not shown).

Image analysis

In vivo tracking of oskMS2‐GFP particles and oskar mRNA distribution within oocytes was performed as previously described (Gaspar et al, 2014). Image segmentation for tracking, colocalization analysis of oskar mRNPs and measurement of bead fluorescence was carried out using a custom particle detector library in ImageJ.

Extraction of NE radial profiles

A section containing a close‐to‐maximal cross section of the nucleus was selected, and the outline of the NE counterstained with WGA was coarsely traced manually. At each point along the outline, the signal under a 5‐μm‐long segment perpendicular to the outline was extracted, resulting in a few hundred to thousand, roughly 2.5‐μm‐long reads on both the cytoplasmic and the nuclear side of the outline. At each point, the position of the NE was determined with sub‐pixel precision through fitting a Gaussian function to the WGA signal. All other recorded signal was positioned relative to the location of the NE and was averaged for a given nucleus. These mean signal intensities were then normalized to the maximum value of a radial profile.

Ex vivo tracking

Ex vivo tracking was done automatically. Tracks displaying linear displacements were manually selected and their directionally manually assigned by overlaying them with the EB1 channel. 8–20% of detected tracks could not be assigned a polarity due to the absence of nearby co‐axial EB1 comets (Fig EV1B). Linear runs were extracted from the detected tracks using a series of custom Excel macros (Gaspar et al, 2014). Runs of unknown polarity were on average shorter than classified minus‐ and plus‐end‐directed runs (Fig EV1D).

Object‐based colocalization

Object‐based colocalization was assayed by measuring the distance between closest‐neighbour objects from the oskar mRNP (reference) and GFP/mKate2 (target) channels within a confined area representing exclusively the nurse cell perinuclear region and cytoplasm. Random colocalization was addressed by seeding the objects of the target channel randomly into the confined area. This simulation was repeated one hundred times to obtain a distribution of expected (random) colocalization. To calculate fraction of colocalization and to compensate for the huge variability of observed particles per image, reference channel objects were randomly assigned into particle clusters representing 160 particles for ex vivo and in situ colocalization analysis, and 100 particles for competitive FISH. With these values, only ~5% of observed objects of the reference channel were excluded from the analysis. Observed colocalization within each cluster was compared to the distribution of simulated random values using one‐sample Student's t‐test (α = 0.01). Significant values were used to calculate the difference between observed and random colocalization to assess true, biological colocalization. These differences were found to be significantly different from zero for all analysed protein molecules in wild‐type samples, except for the negative control Ketel‐GFP (Fig EV4D). By opening the colocalization window (the maximal inter‐neighbour distance), random colocalization rapidly overcomes the observed values (e.g. Fig EV1E–H) due to particle crowdedness both ex vivo and in situ resulting in a probable underestimation of true colocalization. To minimize this effect, the clipping point where the difference was maximal was determined, and the halfway distance between zero and the clipping point was used to compare the effects of different conditions on oskar mRNP composition. This colocalization window was 200 nm ex vivo (non‐fixed specimen), 250 nm in situ and 100 nm STED in situ (fixed).

Temporal colocalization

Temporal colocalization was assayed as described in the legend to Fig EV2A. Importantly, the same microscope settings were used to acquire image sequences of a given protein molecule (e.g. Khc‐mKate2) that allows direct comparison of signal intensities between hetero‐ and homozygous extracts (see thresholding in the legend to Fig EV2A).

Statistical analyses

Transformations and statistical analysis of all the obtained numerical data were carried out in R (Team, 2012) using the R Studio (https://www.rstudio.com/) front‐end and ggplot2 library (Wickham, 2009) to plot the graphs. Normal distribution of the sampled values was determined by Shapiro–Wilk test. Alpha values for statistical tests were chosen based on average sample size as follows: α = 0.05, 1 < N ≤ 10; α = 0.01, 10 < N ≤ 100; and α = 0.001, 100 < N. Sample sizes (pooled from at least two replicates) are indicated in the figures and figure legends. Only two‐sided statistical tests were used. Measurements that were not within the [Q1 (first quartile) − 1.5 × (Q3 (third quartile) −Q1), Q3 + 1.5 × (Q3−Q1)] range of the samples were scored as outliers.

Author contributions

IG and AE conceived the experiments and wrote the manuscript. VS carried out qRT–PCR analysis of mRNAs immunoprecipitated under stringent conditions. AK carried out the EmGFP‐Tm1‐I co‐immunoprecipitation assays. IG carried out the rest of the experiments and data analysis.

Conflict of interest

The authors declare that they have no conflict of interest.

Supporting information

Appendix

Expanded View Figures PDF

Video EV1

Video EV2

Video EV3

Source Data for Appendix

Review Process File

Source Data for Figure 4A

The EMBO Journal (2017) 36: 319–333